The present invention relates to compounds useful as quenchers in a reporter-quencher energy transfer dye pair. Specifically, the present invention relates to non-radiative cyanine quencher compounds, reagents, such as nucleosides/tides and polynucleotides, incorporating such compounds and methods utilizing such compounds and/or reagents.
Nucleic acid hybridization assays comprise an important class of techniques in modern biology. Such assays have diverse applications, including the diagnosis of inherited disease, human identification, identification of microorganisms, paternity testing, virology, and DNA sequencing, e.g., sequencing by hybridization.
An important aspect of nucleic acid hybridization assays is the method used to facilitate detection of the hybridization event. A particularly important class of methods used in nucleic acid hybridization assays employs a reporter-quencher energy-transfer compound pair comprising a xe2x80x9creporterxe2x80x9d compound and a xe2x80x9cquencherxe2x80x9d compound that interact through a fluorescence resonance energy transfer (FRET) process. In these methods, the reporter is a luminescent compound that can be excited either by chemical reaction, producing chemiluminescence, or by light absorption, producing fluorescence. The quencher can interact with the reporter to alter its light emission, usually resulting in the decreased emission efficiency of the reporter. This phenomenon is called quenching. The efficiency of quenching is strongly correlated with the distance between the reporter molecule and the quencher molecule. Thus, in a nucleic acid hybridization assay, detection of a hybridization event is accomplished by designing an energy transfer system in which the spacing between a reporter and a quencher is modulated as a result of the hybridization.
Quencher compounds that are presently used in FRET-based nucleic acid hybridization assays are themselves fluorescent. That is, in addition to quenching the fluorescence of the reporter, the quencher produces fluorescent emissions. This is problematic, particularly in assays employing multiple spectrally resolvable reporters. Because the quencher fluorescence can interfere with the fluorescent signal produced by one or more of the reporters, detection of a hybridization event can be problematic. Accordingly, there remains a continuing need for quencher compounds that are substantially non-fluorescent. In addition, there remains a need for reagents, such as nucleoside/tides and polynucleotides, that incorporate such quencher compounds in order to more reliably monitor, e.g., hybridization events.
The present invention is directed to Applicants"" discovery of a class of non-fluorescent cyanine quencher compounds that are useful in the context of a reporter-quencher energy transfer compound pair. These quencher compounds find particular application in nucleic acid hybridization assays employing fluorescence energy transfer as a means of detection.
In one embodiment, the present invention relates to compounds of formula (I): 
alone or in combination with a counterion thereof, wherein:
p is 0 or 1;
n is 0 or 1;
X is S, Se or O;
N1 is nitrogen;
Z is selected from the group consisting of: 
either:
(a) R2 is A, Y1 is H, and R1 is a linking group, or
(b) R2 is a linking group, and:
(i) R1 is A, or
(ii) p is 1, and R1 and Y1 taken together are (CH2)q;
A is selected from the group consisting of alkyl, aryl, xe2x80x94CH2aryl, and xe2x80x94CH2)mN+(CH3)3;
q is an integer ranging from 2 to 4;
each m is independently an integer ranging from 2 to 12;
A or (CH2)q is unsubstituted or independently substituted with one or more of the same or different xe2x80x94NO2, xe2x80x94OH, alkoxy, xe2x80x94COOH, xe2x80x94COOC1-C4 alkyl, xe2x80x94NHCHO, xe2x80x94NHCOC1-C4 alkyl, xe2x80x94NHCOCH3, xe2x80x94NHCOCH2Cl, xe2x80x94NHCOCHCl2, xe2x80x94NHCOCCl3, xe2x80x94NHCOCF3, xe2x80x94NHCOCH2C6H4-o-NO2, xe2x80x94NHCOCH2OC6H4-o-NO2, xe2x80x94NHCOCH2COCH3, xe2x80x94NHCOCH2xe2x80x94N+C5H5Clxe2x88x92, xe2x80x94NHCOCH2NHCS2CH2C6H5, xe2x80x94NHCOCH2CH2C6H5, xe2x80x94NHCOCH2CH2C6H4-p-OH, xe2x80x94NHCOCH2CH2C6H4-o-NO2, xe2x80x94NHCOC(CH3)2OC6H4-o-NO2, xe2x80x94NHCOC(CH3)2OC6H4-o-Nxe2x95x90NC6H5, xe2x80x94NHCO(CH2)3Cl, xe2x80x94NHCOCH(CH3)2, xe2x80x94NHCOCHxe2x95x90CHC6H4-o-NO2, or xe2x80x94NHCO-2-pyridyl groups; either:
(a) R3, R5 and R6 are H and R4 is xe2x80x94NO2; or
(b) R3 and R4 taken together form a benzo group substituted with one or two xe2x80x94NO2 groups and R5 and R6 are hydrogen; or
(c) R4 and R5 taken together form a benzo group substituted with one or two xe2x80x94NO2 groups and R3 and R6 are hydrogen; or
(d) R5 and R6 taken together form a benzo group substituted with one or two xe2x80x94NO2 groups and R3 and R4 are hydrogen; and with the proviso that when R1 in the compounds of formula (I) has an sp3 hybridized carbon atom that is covalently attached to N1, then that carbon atom is methyl or, when substituted, primary,
and protected derivatives thereof.
The compounds of formula (I) are useful as non-fluorescent quenchers. In addition, they are useful in a composition further comprising a reporter dye, which have utility in determining how well matched are the reporter and quencher dyes with each other with respect to the ability of the quencher to quench the fluorescence of the reporter. The compounds of formula (I) are also useful: (i) when linked to a biomolecule, wherein the biomolecule is also linked to a reporter dye; and (ii) when linked to a biomolecule in a composition with a second biomolecule linked to a reporter dye in detecting the presence of a specific nucleotide sequence in a nucleic acid sample, in detecting the presence of contiguous sequences on a target nucleic acid, in detecting the presence of mutations within a target nucleic acid sequence, in monitoring the kinetics of nucleic acid hybridization, and in monitoring the progression of PCR reactions.
In a second embodiment, the present invention relates to compounds of formula (II) NUCxe2x80x94Lxe2x80x2xe2x80x94R41xe2x80x94Lxe2x80x94D alone or in combination with a counterion thereof, wherein: NUC is a nucleoside, a nucleotide, a nucleoside analog, or a nucleotide analog; Lxe2x80x2 is a bond or a first spacer; R41 is a covalent linkage; L is a bond or a second spacer; and D is the chromophore of a compound of formula (I), defined above, and protected derivatives thereof.
The compounds of formula (II) are useful as monomers for the synthesis of biologically relevant molecules, particularly oligonucleotides, that comprise a non-fluorescent quencher dye.
In a third embodiment, the present invention relates to compounds of the formula (III): 
alone or in combination with a counterion thereof, wherein:
R2-R6, n, p, m, X, Z, NUC, L, Lxe2x80x2 and R41 are defined for formula (I); and
Y1 is H, and protected derivatives thereof.
The compounds of formula (III) are useful as monomers for the synthesis of biologically relevant molecules, particularly oligonucleotides, that comprise a non-fluorescent quencher dye.
In a fourth embodiment, the present invention relates to compounds of formula (IV): 
alone or in combination with a counterion thereof, wherein:
R1, R3-R6, n, p, X, L, Lxe2x80x2, Y1, NUC and R41 are defined as for formulas (I) and (II); and
Z is selected from the group consisting of: 
and protected derivatives thereof.
The compounds of formula (IV) are useful as monomers for the synthesis of biologically relevant molecules, particularly oligonucleotides, that comprise a non-fluorescent quencher dye.
In a fifth embodiment, the present invention relates to compounds of formula (V)
NUCxe2x80x94Cxe2x89xa1Cxe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94NR57xe2x80x94R58xe2x80x94R41xe2x80x94Lxe2x80x94Dxe2x80x83xe2x80x83(V)
alone or in combination with a counterion thereof wherein:
NUC and D are defined as for formula (II);
R41 is selected from a covalent linkage selected from the group consisting of carboxamides, esters, imines, hydrazones, oximes, alkyl amines, thioethers, ethers, thiophenols, aryl amines, boronate esters, hydrazides, N-acylureas or anhydrides, aminotriazines, triazinyl ethers, amidines, ureas, urethanes, thioureas, phosphite esters, silyl ethers, alkyl amines, sulfonamides, and sulfonate esters; a linkage between a pair of specific binding compounds selected from the group consisting of biotin with avidin, biotin with streptavidin, biotin with anti-biotin, IgG with protein A, IgG with protein G, a drug with a drug receptor, a toxin with a toxin receptor, a carbohydrate with a lectin, a carbohydrate with a carbohydrate receptor, a peptide with a peptide receptor; or a linkage between an anionic group and a cationic group; and
L is selected from the group consisting of bonds, alkyldiyls, substituted alkyldiyls, alkylenos, substituted alkylenos, heteroalkyldiyls, substituted heteroalkyldiyls, heteroalkylenos, substituted heteroalkylenos, acyclic heteroatomic bridges, aryldiyls, substituted aryldiyls, arylaryldiyls, substituted arylaryldiyls, arylalkyldiyls, substituted arylalkyldiyls, heteroaryldiyls, substituted heteroaryldiyls, heteroaryl-heteroaryldiyls, substituted heteroaryl-heteroaryldiyls, heteroarylalkyldiyls, substituted heteroarylalkyldiyls, heteroaryl-heteroalkyldiyls, and substituted heteroaryl-heteroalkyldiyls;
R57 is hydrogen or (C1-C6) alkyl; and
R58 is xe2x80x94C(O)xe2x80x94(CH2)rxe2x80x94, xe2x80x94C(O)xe2x80x94CHR59xe2x80x94, xe2x80x94C(O)xe2x80x94Cxe2x89xa1Cxe2x80x94CH2xe2x80x94 or xe2x80x94C(O)xe2x80x94xcfx86xe2x80x94(CH2)rxe2x80x94, where each r is independently an integer from 1 to 5 and xcfx86 is a C6 aryldiyl or a 6-membered heteroaryldiyl, and R59 is hydrogen, (C1-C6) alkyl, a side chain of a gene-encoded amino acid, or a side chain of a non-encoded amino acids,
and protected derivatives thereof.
The compounds of formula (V) are useful as monomers for the synthesis of biologically relevant molecules, particularly oligonucleotides, that comprise a non-fluorescent quencher dye.
In a sixth embodiment, the present invention relates to compounds of formula (VI) 
alone or in combination with a counterion thereof wherein:
R41, L, Lxe2x80x2, and D are defined as for formula (II);
B is a nucleobase or nucleobase analog;
R70 and R71 are each independently xe2x80x94H, xe2x80x94OH or a moiety which blocks polymerase-mediated template-directed polymerization; and
R72 is xe2x80x94OH, or a phosphate ester having the formula 
wherein a is an integer from 0 to 2, or a phosphate ester analog,
and protected derivatives thereof.
In a seventh embodiment, the present invention relates to compounds of formula (VII): 
alone or in combination with a counterion thereof wherein:
R41, L, and D are defined above;
N, O and P represent nitrogen, oxygen and phosphorous, respectively;
Lxe2x80x3 is selected from the group consisting of bonds, alkyldiyls, substituted alkyldiyls, alkylenos, substituted alkylenos, heteroalkyldiyls, substituted heteroalkyldiyls, heteroalkylenos, substituted heteroalkylenos, aryldiyls, substituted aryldiyls, arylaryldiyls, substituted arylaryldiyls, arylalkyldiyls, substituted arylalkyldiyls, heteroaryldiyls, substituted heteroaryldiyls, heteroaryl-heteroaryldiyls, substituted heteroaryl-heteroaryldiyls, heteroarylalkyldiyls, substituted heteroarylalkyldiyls, heteroaryl-heteroalkyldiyls, and substituted heteroaryl-heteroalkyldiyls;
R60 is a phosphite ester protecting group;
R61, when taken alone, is selected from the group consisting of (C1-C6) alkyl, (C1-C6) alkanyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C3-C10) cycloalkyl, (C5-C20) aryl and (C6-C26) arylalkyl, or when taken together with R62 forms a straight-chain or branched (C2-C10) alkyleno or a straight-chain or branched 2-10 membered heteroalkyleno; and
R62, when taken alone, is selected from the group consisting of(C1-C6)alkyl, (C1-C6) alkanyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C3-C10) cycloalkyl, (C5-C20) aryl and (C6-C26) arylalkyl, or when taken together with R61 forms a straight-chain or branched (C2-C10) alkyleno or a straight-chain or branched 2-10 membered heteroalkyleno,
and protected derivatives thereof.
The compounds of formula (VII) are useful as monomers for the chemical synthesis of biologically relevant monomers that comprise a non-fluorescent quencher dye. The compounds of formula (VII) are also useful for labeling biologically relevant molecules themselves.
In an eighth embodiment, the present invention relates to compounds of formula (VIII): 
alone or in combination with a counterion thereof wherein:
N, P, O, R41, L, D, R60, R61, and R62 are defined above;
R63 is hydrogen or an acid-labile hydroxyl protecting group; and
xcexd is an integer from 1 to 30, and protected derivatives thereof
The compounds of formula (VIII) are useful as monomers for the chemical synthesis of biologically relevant monomers that comprise a non-fluorescent quencher dye. The compounds of formula (VIII) are also useful for labeling biologically relevant molecules themselves.
In a ninth embodiment, the present invention relates to a compound of formula (IX): 
alone or in combination with a counterion thereof wherein:
B, R41, L, Lxe2x80x3, D, R60, R61, R62 and R63 are defined as for formula (VII),
and protected derivatives thereof.
The compounds of formula (IX) are useful as monomers for the synthesis of biologically relevant molecules, particularly oligonucleotides, that comprise a non-fluorescent quencher dye.
In a tenth embodiment, the present invention relates to compositions comprising a reporter dye and a quencher dye, wherein the quencher dye is a compound of formula (I). Such compositions have utility in determining how well matched are the reporter and quencher dyes with each other with respect to the ability of the quencher to quench the fluorescence of the reporter, in detecting the presence of a specific nucleotide sequence in a nucleic acid sample, in detecting the presence of contiguous sequences on a target nucleic acid, in detecting the presence of mutations within a target nucleic acid sequence, in monitoring the kinetics of nucleic acid hybridization, and in monitoring the progression of PCR reactions.
In an eleventh embodiment, the present invention relates to an oligonucleotide having attached thereto the chromophore of a compound of formula (I). Such attachment can be via a linking group, described below.
Such an oligonucleotide is useful for detecting the presence of a specific nucleotide sequence in a nucleic acid sample, detecting the presence of contiguous sequences on a target nucleic acid, detecting the presence of mutations within a target nucleic acid sequence, monitoring the kinetics of nucleic acid hybridization, and monitoring the progression of PCR reactions.
In a twelfth embodiment, the present invention relates to an oligonucleotide having attached thereto the chromophore of a reporter dye and the chromophore of a compound of formula (I). Such attachment can be via a linking group, described below. Such oligonucleotides are useful for detecting the presence of a specific nucleotide sequence in a nucleic acid sample, detecting the presence of contiguous sequences on a target nucleic acid, detecting the presence of mutations within a target nucleic acid sequence, monitoring the kinetics of nucleic acid hybridization, and monitoring the progression of PCR reactions.
In a thirteenth embodiment, the present invention relates to a composition comprising a first oligonucleotide having attached thereto the chromophore of a compound of formula (I) and a second oligonucleotide having attached thereto the chromophore of a reporter dye. Such attachments can be via a linking group, described below. Such compositions are useful for detecting the presence of a specific nucleotide sequence in a nucleic acid sample, detecting the presence of contiguous sequences on a target nucleic acid, detecting the presence of mutations within a target nucleic acid sequence, monitoring the kinetics of nucleic acid hybridization, and monitoring the progression of PCR reactions.
In a fourteenth embodiment, the present invention relates to a method for detecting a target nucleic acid sequence in a nucleic acid sample, comprising the steps of:
(a) contacting a target nucleic acid sequence with an oligonucleotide probe, wherein the oligonucleotide probe is labeled with: (1) the chromophore of a compound of formula (I); and (2) a reporter dye;
(b) exposing the oligonucleotide probe to light; and
(c) monitoring the change in fluorescence emission of the reporter dye relative to its emission prior to its contacting the target nucleic acid.
In a fifteenth embodiment, the present invention relates to a method for detecting a target nucleic acid sequence in a nucleic acid sample, comprising the steps of:
(a) contacting a target nucleic acid sequence with a first oligonucleotide probe having attached thereto the chromophore of the compound of formula (I); (b) contacting a target nucleic acid sequence with a second oligonucleotide probe having attached thereto a reporter dye;
(b) exposing the oligonucleotide probes to light; and
(c) monitoring the change in fluorescence emission of the reporter dye relative to its emission prior to its contacting the target nucleic acid.
These and other objects, features, and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.